HybridsHybridization is the formation of organisms by cross-fertilization between individuals of different species, which is key to the evolution and diversification of 30-70% flowering plants. Hybrids generally express phenotypes intermediate to those of parents, which sometimes result in heterosis or hybrid vigor such that the hybrids survive better than both parents, but the opposite is also possible. Studies comparing the metabolite expression of hybrids with that of their parent species are limited, but existing evidence suggests that hybrids may express more diverse combinations of secondary metabolites or produce unique phytochemicals novel to both parental species. Hybridization in the species rich Nepenthaceae is extensive, either naturally or artificially driven by high horticultural demand as ornamental plants.
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According to Harry James Veitch in 1906 (Veitch, 1979), the first Nepenthes artificial hybridization was initiated by John Dominy who produced N. × dominiana from N. rafflesiana with an unnamed species in 1862, followed by N. × hybrida from N. khasiana with an unnamed Bornean species in 1866.
Despite the long history, studies on Nepenthes hybrids and their chemical compositions are generally limited. There is no study to date on the metabolite expression of hybrids in this family. We performed a non-targeted metabolomics analysis of the pitchers of two Nepenthes species with different dietary habits, namely the omnivorous N. ampullaria and carnivorous N. rafflesiana with their hybrid (N. × hookeriana) for a comparative study.
The current study aims to unveil the effects of hybridization in pitcher plants based on comparative metabolic profiling through an untargeted approach for identifying metabolites. Liquid chromatography-mass spectrometry (LC-MS) is used to classify and compare chemical profiles between N. × hookeriana, a natural hybrid with its parents, N. ampullaria and N. rafflesiana.
Despite the long history, studies on Nepenthes hybrids and their chemical compositions are generally limited. There is no study to date on the metabolite expression of hybrids in this family. We performed a non-targeted metabolomics analysis of the pitchers of two Nepenthes species with different dietary habits, namely the omnivorous N. ampullaria and carnivorous N. rafflesiana with their hybrid (N. × hookeriana) for a comparative study.
The current study aims to unveil the effects of hybridization in pitcher plants based on comparative metabolic profiling through an untargeted approach for identifying metabolites. Liquid chromatography-mass spectrometry (LC-MS) is used to classify and compare chemical profiles between N. × hookeriana, a natural hybrid with its parents, N. ampullaria and N. rafflesiana.
Timeline of Pitcher DevelopmentPitcher tissues of closely related lowland Nepenthes species, N. ampullaria Jack and N. rafflesiana Jack with their hybrid N. × hookeriana Lindl., all of which were originally transplanted from Endau-Rompin National Park, were sampled from an experimental terrace at Universiti Kebangsaan Malaysia (2°55'12.7"N, 101°46'59.7"E).
For the sampling of pitchers, daily observations of morphological changes were performed in situ throughout pitcher development. The three species showed different pitcher longevity with N. ampullaria pitchers being the most long-lived (> six months) compared to the short-lived (≤ two months) N. rafflesiana pitchers with the hybrid pitchers showing intermediate longevity (2-3 months). It took around 1 month (22-33 days) from pitcher initiation at the tendril tip to pitcher opening for all three species. Pitcher development continues after opening and mature within seven days with a stable morphology, namely the positioning of the lid, peristome structure, coloration, and pitcher size. Therefore, seven-day-old mature pitchers after lid opening were chosen to standardize sampling for metabolomics analysis. |
LC-MS/MS Metabolite ProfilingThe sampling was performed in the morning between 0900-1100 hours during October to December 2015 with at least four biological replicates from each species (Rosli et al., 2018). The whole-pitcher samples excluding the tendril were ground until fine homogenous powders using pestle and mortar pre-chilled with liquid nitrogen, collected in a Falcon tube, and freeze-dried for 48 hours. Dried powder samples (100 mg) were extracted using 200 µL of methanol:chloroform:water (3:1:1) via sonication-assisted method.
A total of 1,441 metabolite features were profiled from the three species in which 43.3% of features in the hybrid samples were not found in either of its parents. The partial least squares discriminant analysis (PLS-DA) found 324 metabolite features with variable in projection (VIP) values greater than one in which 55 features were statistically significant. |
A total of 223 metabolites were putatively identified with manual searches using public metabolite databases. Phenol group was found to be the most abundant secondary metabolites due to a high flavonoid content, especially in N. rafflesiana. The most abundant feature 476.3s:449.102, putatively identified as astragalin, was found to be the top feature in the VIP analysis for distinguishing between the three species as a chemical marker.
Significance
This is the first study comparing metabolites in the carnivory organs of different Nepenthes species with comprehensive profiling and putative identification. The differential metabolite compositions in the pitchers of different species might have ecological implications with the hybrid showing intermediate phenotype between the parents as well as manifesting unique metabolites. Subsequently, compounds with the bioactivity of interest can be targeted for isolation and further characterization
References
Book
Grant
- Rosli MAF, Mediani A, Azizan KA, Baharum SN & Goh H-H* (2021) UPLC-TOF-MS/MS-based metabolomics analysis reveals species-specific metabolite compositions in pitchers of Nepenthes ampullaria, Nepenthes rafflesiana, and their hybrid Nepenthes × hookeriana. Front. Plant Sci. 12:655004. doi: 10.3389/fpls.2021.655004
- Rosli MA, Azizan, KA & Goh H-H* (2018) Antioxidant activity of pitcher extracts from three Nepenthes species. Sains Malaysiana 47(12):3069-3075.
- Rosli MA, Azizan, KA, Baharum SN & Goh H-H* (2017) Mass Spectrometry Data of Metabolomics Analysis of Nepenthes Pitchers. Data in Brief 14:295-297.
Book
- Rosli A.F., Baharum S. N. & Goh H-H. (2019) Merungkai kandungan fitokimia dalam kendi periuk kera (Nepenthes sp.). UKM Press ISBN 9789672388296
Grant
- PROTEOMIC AND METABOLOMIC SCREENING OF DIFFERENT NEPENTHES SPECIES - Research University Grant: DIP-2014-008 [1 Aug 2014 – 31 Jul 2017]
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